Metabolic regulation of the squid nerve Na(+)/Ca (2+) exchanger: recent developments.

In squid nerves, MgATP modulation of the Na(+)/Ca(2+) exchanger requires the presence of a cytosolic protein which becomes phosphorylated during the process. This factor has been recently identified. Mass spectroscopy and Western blot analysis established that it is a member of the lipocalin superfamily of lipid-binding proteins (LBP or FABP) of 132 amino acids. We called it regulatory protein of squid nerve sodium/calcium exchanger (ReP1-NCXSQ, access to GenBank EU981897).ReP1-NCXSQ was cloned, expressed, and purified. Circular dichroism, far-UV, and infrared spectroscopy suggest a secondary structure, predominantly of beta-sheets. The tertiary structure prediction provides ten beta-sheets and two alpha-helices, characteristic of most of LPB. Functional experiments showed that, to be active, ReP1-NCXSQ must be phosphorylated by MgATP, through the action of a kinase present in the plasma membrane. Moreover, PO4-ReP1-NCXSQ can stimulate the exchanger in the absence of ATP. An additional crucial observation was that, in proteoliposomes containing only the purified Na(+)/Ca(2+) exchanger, PO4-ReP1-NCXSQ promotes activation; therefore, this upregulation has no other requirement than a lipid membrane and the incorporated exchanger protein.Recently, we solved the crystal structure of ReP1-NCXSQ which was as predicted: a "barrel" consisting of ten beta-sheets and two alpha-helices. Inside the barrel is the fatty acid coordinated by hydrogen bonds with Arg126 and Tyr128. Point mutations showed that neither Tyr20Ala, Arg58Val, Ser99Ala, nor Arg126Val is necessary for protein phosphorylation or activity. On the other hand, Tyr128 is essential for activity but not for phosphorylation. We can conclude that (1) for the first time, a role of an LBP is demonstrated in the metabolic regulation of an ion exchanger; (2) phosphorylation of this LBP can be separated from the activation capacity; and (3) Tyr128, a candidate to coordinate lipid binding inside the barrel, is essential for activity.

Metabolic regulation of the squid nerve Na⁺/Ca²âº exchanger: recent kinetic, biochemical and structural developments.

The Na⁺/Ca²âº exchangers are structural membrane proteins, essential for the extrusion of Ca²âº from most animal cells. Apart from the transport sites, they have several interacting ionic and metabolic sites located at the intracellular loop of the exchanger protein. One of these, the intracellular Ca²âº regulatory sites, are essential and must be occupied by Ca²âº to allow any type of ion (Na⁺ or Ca²âº) translocation. Intracellular protons and Na⁺ are inhibitory by reducing the affinity of the regulatory sites for Ca²âº; MgATP stimulates by antagonizing H⁺ and Na⁺. We have proposed a kinetic scheme to explain all ionic and metabolic regulation of the squid nerve Na⁺/Ca²âº exchanger. This model uniquely accounts for most of the new kinetic data provided here; however, none of the existing models can explain the trans effects of the Ca(i)²âº-regulatory sites on external cation transport sites; i.e. all models are incomplete. MgATP up-regulation of the squid Na⁺/Ca²âº exchanger requires a cytosolic protein, which has been recently identified as a member of the lipocalin super family of Lipid Binding Proteins (LBP or FABP) of 132 amino acids (ReP1-NCXSQ, access to GenBank EU981897). This protein was cloned, expressed and purified. To be active, ReP1-NCXSQ must be phosphorylated from MgATP by a kinase present in the plasma membrane. Phosphorylated ReP1-NCXSQ can stimulate the exchanger in the absence of ATP. Experiments with proteoliposomes proved that this up-regulation can take place just with the lipid membrane and the exchanger protein. The structure of ReP1-NCXSQ predicted from the amino acid sequence has been confirmed by X-ray crystal analysis; it has a "barrel" formed by ten beta sheets and two alpha helices, with a lipid coordinated by hydrogen bonds with Arg 126 and Tyr 128.

Optimal metabolic regulation of the mammalian heart Na(+)/Ca(2+) exchanger requires a spacial arrangements with a PtdIns(4)-5kinase.

In inside-out bovine heart sarcolemmal vesicles, p-chloromercuribenzenesulfonate (PCMBS) and n-ethylmaleimide (NEM) fully inhibited MgATP up-regulation of the Na(+)/Ca(2+) exchanger (NCX1) and abolished the MgATP-dependent PtdIns-4,5P2 increase in the NCX1-PtdIns-4,5P2 complex; in addition, these compounds markedly reduced the activity of the PtdIns(4)-5kinase. After PCMBS or NEM treatment, addition of dithiothreitol (DTT) restored a large fraction of the MgATP stimulation of the exchange fluxes and almost fully restored PtdIns(4)-5kinase activity; however, in contrast to PCMBS, the effects of NEM did not seem related to the alkylation of protein SH groups. By itself DTT had no effect on the synthesis of PtdIns-4,5P2 but affected MgATP stimulation of NCX1: moderate inhibition at 1mM MgATP and 1µM Ca(2+) and full inhibition at 0.25mM MgATP and 0.2µM Ca(2+). In addition, DDT prevented coimmunoprecipitation of NCX1 and PtdIns(4)-5kinase. These results indicate that, for a proper MgATP up-regulation of NCX1, the enzyme responsible for PtdIns-4,5P2 synthesis must be (i) functionally competent and (ii) set in the NCX1 microenvironment closely associated to the exchanger. This kind of supramolecular structure is needed to optimize binding of the newly synthesized PtdIns-4,5P2 to its target region in the exchanger protein.

Key role of a PtdIns-4,5P2 micro domain in ionic regulation of the mammalian heart Na+/Ca2+ exchanger.

Phosphatidylinositol biphosphate (PtdIns-4,5P(2)) plays a key role in the regulation of the mammalian heart Na(+)/Ca(2+) exchanger (NCX1) by protecting the intracellular Ca(2+) regulatory site against H(+)(i) and (H(+)(i)+Na(+)(i)) synergic inhibition. MgATP and MgATP-gamma-S up-regulation of NCX1 takes place via the production of this phosphoinositide. In microsomes containing PtdIns-4,5P(2) incubated in the absence of MgATP and at normal [Na(+)](i), alkalinization increases the affinity for Ca(2+)(i) to the values seen in the presence of the nucleotide at normal pH; under this condition, addition of MgATP does not increase the affinity for Ca(2+)(i) any further. On the other hand, prevention of Na(+)(i) inhibition by alkalinization in the absence of MgATP does not take place when the microsomes are depleted of PtdIns-4,5P(2). Experiments on NCX1-PtdIns-4,5P(2) cross-coimmunoprecipitation show that the relevant PtdIns-4,5P(2) is not the overall membrane component but specifically that tightly attached to NCX1. Consequently, the highest affinity of the Ca(2+)(i) regulatory site is seen in the deprotonated and PtdIns-4,5P(2)-bound NCX1. Confirming these results, a PtdIns-5-kinase also cross-coimmunoprecipitates with NCX1 without losing its functional competence. These observations indicate, for the first time, the existence of a PtdIns-5-kinase in the NCX1 microdomain.

Squid nerve Na+/Ca2+ exchanger expressed in Saccharomyces cerevisiae: up-regulation by a phosphorylated cytosolic protein (ReP1-NCXSQ) is identical to that of native exchanger in situ.

This work shows, for the first time, a properly metabolically regulated squid nerve Na(+)/Ca(2+) exchanger (NCXSQ1) heterologous expressed in Saccharomyces cerevisiae. The exchanger was fused to the enhanced green fluorescence protein (eGFP) on its C-terminus and had two tags, a Strep-tag II and 6 histidines, added to the N-terminal region (ST-6HB-NCXSQ1-eGFP). The eGFP fluorescence signal co-localized with that of the plasma membrane marker FM1-43 in whole cells that displayed an uptake of Ca(2+) with the expected characteristics of the reverse Na(+)/Ca(2+) exchange mode. Similar to squid nerve membrane vesicles, inside-out yeast plasma membrane vesicles (ISOV) showed a Ca(2+)(i) regulation of the forward mode that was modulated by previously phosphorylated regulatory cytosolic protein (ReP1-NCXSQ). On the other hand, a close association between NCXSQ1 and ReP1-NCXSQ, estimated by co-immunoprecipitation, was independent of ReP1-NCXSQ phosphorylation. An additional crucial observation was that in proteoliposomes containing only the ST-6HB-NCXSQ1-eGFP protein, Na(+)/Ca(2+) exchange was stimulated by phosphorylated ReP1-NCXSQ; i.e., this up-regulation needs no other requirement besides the lipid membrane and the exchanger protein. Finally, this work provides a potential approach to obtain enough purified NCXSQ1 for structural and biochemical studies which have been delayed due to the lack of sufficient material.

A novel lipid binding protein is a factor required for MgATP stimulation of the squid nerve Na+/Ca2+ exchanger.

Here we identify a cytosolic factor essential for MgATP up-regulation of the squid nerve Na(+)/Ca(2+) exchanger. Mass spectroscopy and Western blot analysis established that this factor is a member of the lipocalin super family of lipid binding proteins of 132 amino acids in length. We named it Regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ). ReP-1-NCXSQ was cloned, over expressed and purified. Far-UV circular dichroism and infrared spectra suggest a majority of beta-strand in the secondary structure. Moreover, the predicted tertiary structure indicates ten beta-sheets and two short alpha-helices characteristic of most lipid binding proteins. Functional experiments showed that in order to be active ReP1-NCXSQ must become phosphorylated in the presence of MgATP by a kinase that is Staurosporin insensitive. Even more, the phosphorylated ReP1-NCXSQ is able to stimulate the exchanger in the absence of ATP. In addition to the identification of a new member of the lipid binding protein family, this work shows, for the first time, the requirement of a lipid binding protein for metabolic regulation of an ion transporting system.

Some biochemical properties of the upregulation of the squid nerve Na+/Ca2+ exchanger by MgATP and phosphoarginine.

In squid nerve MgATP upregulation of Na+/Ca2+ exchange requires a soluble cytosolic regulatory protein (SCRP) of about 13 kDa; phosphoarginine (PA) stimulation does not. MgATP-gamma-S mimics MgATP. When a 30-10-kDa cytosolic fraction is exposed to 0.5 mM [32P]ATP in the same solution used for transport assays, and in the presence of native membrane vesicles, a 13-kDa and a 25-kDa band become phosphorylated. Membrane vesicles alone do not show these phosphorylated bands and heat denaturation of the cytosolic fraction prevents phosphorylation. Moreover, staurosporine, a general inhibitor of kinases, does not affect MgATP + SCRP stimulation of the exchanger or the phosphorylation of the 13 kDa but prevents phosphorylation of the 25-kDa cytosolic band. The 30-10-kDa fraction phosphorylated in the presence of staurosporine stimulates Na+/Ca2+ exchange in vesicles in the absence of ATP but with Mg2+ in the medium. The 30-10-kDa fraction is not phosphorylated by PA. In membrane vesicles two protein bands, at about 60 kDa and 70 kDa identified as the low molecular weight neurofilament (NF), are phosphorylated by PA, but not by MgATP. This phosphorylation is specific for PA, insensitive to staurosporine (similar to the PA-stimulated fluxes), and labile. In addition, co-immunoprecipitation was observed between the NF and the exchanger protein. Under the conditions of these experiments no phosphorylation of the exchanger is detected, either with MgATP or PA.

In bovine heart Na+/Ca2+ exchanger maximal Ca2+i affinity requires simultaneously high pHi and PtdIns-4,5-P2 binding to the carrier.

Na+ i-dependent Ca2+ uptake, Na+-dependent Ca2+ release, and PtdIns-4,5-P2 binding to Na+/Ca2+ exchanger (NCX1) as a function of extravesicular (intracellular) [Ca2+] were measured. Alkalinization increases Ca2+ i affinity and PtdIns-4,5-P2 bound to NCX1; these effects are abolished by pretreatment with PtdIns-PLC and are insensitive to MgATP. Acidification reduces Ca2+ i affinity. MgATP reverts it only partially despite the fact that the PtdIns-4,5-P2 bound to NCX1 reaches the same levels as at pH 7.8. Extravesicular Na+-stimulated and Ca2+-dependent Ca2+ efflux indicate the Ca2+ regulatory site is involved. Therefore, to display maximal affinity to Ca2+ i, PtdIns-4,5-P2 binding and deprotonation of NCX1 are simultaneously need.

Maximal Ca2+i stimulation of cardiac Na+/Ca2+ exchange requires simultaneous alkalinization and binding of PtdIns-4,5-P2 to the exchanger.

Using bovine heart sarcolemma vesicles we studied the effects of protons and phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) on the affinity of the mammalian Na(+)/Ca(2+) exchanger (NCX1) for intracellular Ca(2+). By following the effects of extravesicular ligands in inside-out vesicles, their interactions with sites of NCX1 facing the intracellular medium were investigated. Two Na(+)-gradient-dependent fluxes were studied: Ca(2+) uptake and Ca(2+) release. PtdIns-4,5-P2 binding to NCX1 was investigated in parallel. Without MgATP (no 'de novo' synthesis of PtdIns-4,5-P2), alkalinization increased the affinity for Ca(2+) and the PtdIns-4,5-P2 bound to NCX1. Vesicles depleted of phosphoinositides were insensitive to alkalinization, but became responsive following addition of exogenous PtdIns-4,5-P2 or PtdIns plus MgATP. Acidification reduced the affinity for Ca(2+)(ev); this was only partially reversed by MgATP, despite the increase in bound PtdIns-4,5-P2 to levels observed with alkalinization. Inhibition of Ca(2+) uptake by increasing extravesicular [Na(+)] indicates that it is related to H(+)(i) and Na(+)(i) synergistic inhibition of the Ca(2+)(i) regulatory site. Therefore, the affinity of the NCX1 Ca(2+)(i) regulatory site for Ca(2+) was maximal when both intracellular alkalinization and an increase in PtdIns-4,5-P2 bound to NCX1 (not just of the total membrane PtdIns-4,5-P2) occurred simultaneously. In addition, protons influenced the distribution, or the exposure, of PtdIns-4,5-P2 molecules in the surroundings and/or on the exchanger protein.

Phosphoarginine regulation of the squid nerve Na+/Ca2+ exchanger: metabolic pathway and exchanger-ligand interactions different from those seen with ATP.

In squid nerves the Na(+)-Ca(2+) exchanger is up-regulated by ATP and phosphoarginine (PA). ATP regulation involves drastic alterations in the Na(+)(i), H(+)(i) and Ca(2+)(i) interactions with the large intracellular cytoplasmic loop of the exchanger protein. In this work we explored the mechanisms associated with PA regulation in intracellular dialysed squid axons and squid optic nerve membrane vesicles. Dialysed axons were used to measure the four modes of exchange fluxes (Na(+)(o)-Ca(2+)(i) or forward exchange, Ca(2+)(o)-Na(+)(i) or reverse exchange, Ca(2+)(o)-Ca(2+)(i) exchange and Na(+)(o)-Na(+)(i) exchange) under controlled intra- and extracellular conditions. Inside-out membrane vesicles allowed measurement of the Na(+)-gradient-dependent (45)Ca(2+) uptake (forward mode) as influenced by ligands and digestion with chymotrypsin from the intracellular side. The results show that, unlike ATP, PA regulation does not affect the H(+)(i), Na(+)(i) and Ca(2+)(i) interactions with the intracellular 'regulatory' loop, but increases the affinity of the intracellular transport sites, preferentially for Ca(2+)(i) (about 20-fold) over Na(+)(i) (50%); i.e. PA favours the forward mode over the other exchange modes. Intracellular chymotrypsin digestion removed ATP regulation while leaving modulation by PA unmodified. Western blot analysis suggested that chymotrypsin disrupts the large intracellular loop. Together these results indicate that ATP and PA regulations are associated with different structures inside and outside the exchanger protein. Based on these observations we expanded our previous model for metabolic regulation of the Na(+)-Ca(2+) exchanger by adding to the original 'ATP region' a new zone, the 'PA region', related to the intracellular transport sites for Na(+)(i) and Ca(2+)(i). This new model is able to explain most previous and present results.

Regulation of phosphatidylinositol-4,5-biphosphate bound to the bovine cardiac Na+/Ca2+ exchanger.

Western blot and cross immunoprecipitation analysis with specific antibodies demonstrate that in bovine heart sarcolemmal vesicles phosphatidylinositol-4,5-biphosphate (PtdIns-4,5-P(2)) binds strongly to the Na(+)/Ca(2+) exchanger (NCX1). This binding is modulated by ATP, Ca(2+), vanadate, exchanger inhibitory peptide (XIP), and PLC-PtdIns specific in a way resembling the ATP regulation of the exchange fluxes. With 1 microM Ca(2+), 3 mM Mg(2+), and 0.4 mM vanadate, 1 mM ATP increased about twofold the bound PtdIns-4,5-P(2), reaching a steady state in 3-5 s at 37 degrees C. With 100 microM Ca(2+), ATP had no effect on the PtdIns-4,5-P(2) bound to NCX1 or on the exchange fluxes. Without vanadate the bound PtdIns-4,5-P(2) was largely reduced; under this condition ATP failed to increase it and did not stimulate the exchanger. XIP inhibits the exchanger, more noticeable in the absence of ATP. With XIP, ATP does not modify the levels of bound PtdIns-4,5-P(2); however there is a small but distinct ATP stimulation of the exchanger. Vesicles pretreated with PtdIns-PLC, showed no de novo, [(32)P]ATP-induced, production of PtdIns-4,5-P(2), but some ATP-stimulated increase in the bound PtdIns-4,5-P(2) was detected; however, that increase did not exceed the levels found with vanadate and no ATP. These results indicate that in bovine heart sarcolemmal vesicles, ATP upregulation of NCX1 is related to PtdIns-4,5-P(2) bound to the exchanger, perhaps over a "threshold" or "unspecific" amount. In addition, vanadate could influence the amount of detected PtdIns-4,5-P(2) either by inhibiting phosphoinositide-specific phosphatases and/or by inducing a redistribution of PtdIns-4,5-P(2) molecules associated with the Na(+)/Ca(2+) exchanger.

MgATP and phosphoinositides activate Na(+)/Ca(2+) exchange in bovine brain vesicles. Comparison with other Na(+)/Ca(2+) exchangers.

We investigated the metabolic modulation of the Na(+)/Ca(2+) exchanger in membrane vesicles obtained from bovine brain. The Na(+)/Ca(2+) exchanger was activated by MgATP with a K(0.5) of 336 micro M. Unlike the squid nerve Na(+)/Ca(2+) exchanger, this effect required no cytosolic component. Also, stimulation is the same in vesicles prepared and/or assayed at the ionic strength found in mammals (160 mM) or marine animals (300 mM). Other differences between squid and bovine nerve are that the bovine brain Na(+)/Ca(2+) exchanger is not stimulated by phosphagens, either phosphoarginine (molluscan source) or phosphocreatine (mammalian source); and that stimulation by MgATP in bovine brain is related to the production of polyphosphatidylinositides. In this regard bovine heart and brain Na(+)/Ca(2+) exchangers behave similarly. These results indicate that the mechanisms of metabolic regulation of the squid and mammalian nerve Na(+)/Ca(2+) exchangers are not alike and represent differences between species. Some differences found between bovine heart and brain exchangers, such as MgATP stimulation even at saturating [Ca(2+)] and the smaller degree of activation by adenosine 5'- O-(3-thiotriphosphate) (ATP-gamma-S) in the brain, may be related to the unequal isoform population in both tissues.